A novel cell-based high-throughput assay for ALS

描述（由申请人提供）：超氧化物歧化酶-1（SOD 1）突变导致家族性肌萎缩侧索硬化症（ALS）。在嵌合或条件突变小鼠中的研究表明，非神经元细胞在ALS相关的神经变性中起重要作用。我们已经发现用突变体SOD 1星形胶质细胞调节的培养基在体外杀死野生型胚胎干细胞衍生的运动神经元（ES-MN）。这个为期2年的翻译R21旨在确定用于治疗ALS的神经保护剂。我们已经成功地验证了ALS的ES-MN细胞模型，并获得了所有必要的专业知识，设备和化学库，以开展高通量筛选（HTS）工作。我们将开始完成我们的检测试剂盒的优化和验证。然后，我们将分四步执行HTS。首先，为了微调测定参数，我们将筛选2，000种生物活性化合物的小集合，以确定它们在SOD 1星形胶质细胞条件培养基存在下增强ES-MN存活的能力。第二，然后在单一浓度（10 5 M）和一个参数（ES-MN存活率）下逐一筛选约80，000种化合物。第三，将通过以下方式对命中进行验证：（a）多次重复检测;（B）重新合成/回购后重新检测;以及（c）绘制涵盖初始筛选中使用的浓度的剂量-反应曲线。第四，我们收集的结构相关的化合物将与经验证的命中平行测试，以确认每类化合物的生物活性，并选择最有效的化合物。先导化合物定义为具有可重现神经保护活性的化合物，效力为EC 50 d 5 5 M，最大效应为阳性对照的75%。与此计划公告的意图一致，我们的建议提供了一系列研究，旨在开发一种用于筛选治疗药物的检测方法。我们预计，到这个项目结束时，我们的整个小分子库将被测试，并且将选择约100个命中，直接导致ALS的治疗开发项目。后者将包括基于体外二次筛选的临床前研究，一组体内毒性和药代动力学研究，以及在表达突变SOD 1的转基因小鼠中测试主要候选药物。 公共卫生相关性：肌萎缩侧索硬化症（ALS）是一种常见的致命性麻痹性疾病，影响成年人，没有有效的治疗。我们最近确定并验证了在实验室培养皿中复制ALS的方法-这一突破导致了这种疾病的功能性细胞模型的发展。通过将这些新的细胞模型与高通量技术相结合，我们将在短时间内筛选大量的小分子，最终目标是发现对ALS具有神经保护活性的化合物。

DESCRIPTION (provided by applicant): Mutations in superoxide dismutase-1 (SOD1) cause familial amyotrophic lateral sclerosis (ALS). Studies in chimeric or conditional mutant mice indicate that non- neuronal cells play an important role in ALS-related neurodegeneration. We have found that culture medium conditioned with mutant SOD1 astrocytes kills wild- type embryonic stem cell-derived motor neurons (ES-MNs) in vitro. This 2-year translational R21 aims at identifying neuroprotective agents for the treatment of ALS. We have succeeded in miniaturizing our ES-MNs cell model of ALS and have secured all of the necessary expertise, equipment, and chemical libraries to embark on a high-throughput screening (HTS) effort. We will begin by completing the optimization and validation of our assay. We will then perform our HTS in four steps. First, to fine-tune the assay parameters, we will screen a small collection of 2,000 biologically active compounds for their ability to enhance ES-MN survival in the presence of SOD1 astrocyte-conditioned medium. Second, a collection of ~80,000 compounds will then be screened one-by-one at a single concentration (10 5M) and with one parameter (ES-MN survival). Third, hits will be validated by: (a) repeat testing in multiple replicates; (b) retesting after resynthesis/repurchase; and (c) performing dose-response curves spanning the concentrations used in the initial screening. Fourth, structurally-related compounds from our collection will be tested in parallel with validated hits to confirm the bioactivity of each class of compounds, and to select the most potent. Leads will be defined as compounds with a reproducible neuroprotective activity, a potency of EC50 d 5 5M and a maximum effect e 75% of that of the positive control. In agreement with the intent of this program announcement, our proposal offers a set of investigations geared toward developing an assay for the screening of therapeutics. We anticipate that, by the end of this project, our entire library of small molecules will have been tested and that ~100 hits will have been selected, leading directly into a therapy development project for ALS. The latter will consist of preclinical studies based on in vitro secondary screens, a set of in vivo toxicity and pharmacokinetic investigations, and testing of the lead candidates in transgenic mice expressing mutant SOD1. Public Health Relevance: Amyotrophic lateral sclerosis (ALS) is a common fatal paralytic disorder affecting adults with no effective treatment. We have recently identified and validated a means of reproducing ALS in a laboratory dish - a breakthrough which has led to the development of functional cell models of this disease. By using these new cell models in conjunction with high- throughput technologies, we will screen large numbers of small molecules in a brief period of time with the ultimate goal of discovering compounds with neuroprotective activities against ALS.